US4617262A - Assaying for circulating immune complexes with labeled protein A - Google Patents
Assaying for circulating immune complexes with labeled protein A Download PDFInfo
- Publication number
- US4617262A US4617262A US06/516,465 US51646583A US4617262A US 4617262 A US4617262 A US 4617262A US 51646583 A US51646583 A US 51646583A US 4617262 A US4617262 A US 4617262A
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- US
- United States
- Prior art keywords
- cic
- complex
- polyethylene glycol
- label
- spa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/967—Standards, controls, materials, e.g. validation studies, buffer systems
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/971—Capture of complex after antigen-antibody reaction
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/828—Protein A
Definitions
- This invention relates to methods of analyzing for circulating immune complexes in mammalian blood serum and more particularly to methods of analyzing for circulating immune complexes using an enzyme-linked immunoassay.
- Macromolecular circulating immune complexes are formed in the blood serum of mammals by the interaction of endogenous or exogenous antigens and specific antibodies formed against these antigens by the host.
- Typical exogenous antigens which provoke the formation of antibodies include antigens associated with infectious agents such as viruses, bacteria, parasites and fungi, as well as drugs, foods, and allergens.
- Typical endogenous antigens include cellular antigens such as ribosomes, nucleic acid, nucleoproteins, cell surface antigens, mitochondria and the like, and plasma factors such as rheumatoid factor, thyroglobulin, and tumor-associated antigens (e.g., carcinoembryonic antigen) and the like.
- cellular antigens such as ribosomes, nucleic acid, nucleoproteins, cell surface antigens, mitochondria and the like
- plasma factors such as rheumatoid factor, thyroglobulin, and tumor-associated antigens (e.g., carcinoembryonic antigen) and the like.
- tumor-associated antigens e.g., carcinoembryonic antigen
- the processing of immune complexes by the host can also result in immunopathology such as acute and chronic glomerulonephritis, vasculitis, pancreatitis, hepatitis, serum sickness, and the like, in both man and other mammals.
- immunopathology such as acute and chronic glomerulonephritis, vasculitis, pancreatitis, hepatitis, serum sickness, and the like, in both man and other mammals.
- a number of methods of analyzing for CIC have been developed. Physical methods such as analytical ultracentrifugation, gel filtration, sucrose density gradient centrifugation, ultrafiltration, electrophoresis and nephelometry have been used. Chemical methods such as precipitation with polyethylene glycol and cryoprecipitation have also been used. Cellular methods depending on the interaction of CIC with Fc-receptors (FcR) in the cell membrane, such as platelet aggregation, Raji cell assay, rosette inhibition, release of soluble enzymes by eosinophils and basophils, and the bull sperm agglutination assay, have been used.
- FcR Fc-receptors
- Immunologic detection methods for CIC include competitive assays, complement dependent assays, antiglobulin methods and binding to staphylococcal protein-A. A review of prior assay methods for CIC is found in Ritzmann, S. E., and Daniels, J. C., Clinical Chemistry 28(6), 1259-1271 (1982).
- Hallgren, R., et al., Ann. rheum. Dis. 35, 306-313 (1976) disclose assaying CIC by first separating CIC from the other proteins in the serum by exclusion chromatography on a polysaccharide gel and then precipitating with radiolabeled staphylococcal protein-A in the presence of polyethylene glycol. The excess reagent is separated and the radioactivity of the precipitated CIC is measured by counting in the usual way.
- Faiferman I., et al., Arthritis and Rheumatism 25 (7), 799-801 (1982) disclose a fluoroimmunoassay for CIC wherein the complexes are first selectively precipitated with a 2.5% solution of polyethylene glycol, then resuspended and contacted with a solution of SPA linked to a fluorescent label, whereby a complex of CIC and fluorescent labeled SPA is formed. The complex is again precipitated with polyethylene glycol, the excess reagent is removed, and the fluorescence of the bound reagent is measured with a spectrofluorometer. Faiferman et al. disclose that the label used on the SPA could also be an enzyme label.
- a further object is to provide a method for assaying CIC in mammalian serum by means of a label immunoassay.
- a further object is to provide a method for assaying CIC in mammalian serum by means of an enzyme-linked immunoassay.
- a further object is to provide a method for assaying CIC using staphylococcal protein-A which does not require a preliminary separation of CIC from uncomplexed immunoglobulins.
- a further object is to provide an immunoassay for CIC in mammalian serum having a single precipitation step.
- a further object is to provide an analytical reagent specially adapted for an immunoassay for CIC in mammalian serum having a single precipitation step.
- the assay of this invention is based upon the discovery that SPA binds preferentially to CIC.
- This preferential binding affinity allows conditions to be selected wherein the CIC can be bound to SPA and precipitated without preliminary separation of CIC from uncomplexed immunoglobulins. Accordingly, it is possible to eliminate the preliminary separation step used in assays of the prior art and conduct the assay in a single tube with a single precipitation step. This materially simplifies the assay and is therfore a substantial improvement over the known assays for CIC.
- the serum containing CIC and uncomplexed immunoglobulins is mixed with a solution containing SPA labeled with a suitable label such as an enzyme, a radioactive label or a fluorescent label and incubated for a period of time sufficient to form a CIC-SPA-label complex.
- a suitable label such as an enzyme, a radioactive label or a fluorescent label
- the conditions may be chosen so that the CIC-SPA-label complex is formed while no substantial amount of complex between labeled SPA and uncomplexed immunoglobulins is formed.
- the conditions favorable for this result are a temperature between about 18° C. and about 25° C., preferably about 20° C., an amount of SPA-label in excess of that required for binding all the CIC, and an incubation time sufficient for the completion of complex formation. It has been found that the reaction is essentially complete in about 15 minutes.
- the CIC-SPA-label complex is then precipitated by addition of a solution, preferably an aqueous solution, of polyethylene glycol, to produce a final concentration of PEG in the analytical solution of between 2.0% and 3.5%, by weight.
- concentration of PEG in the analytical solution is about 3.5% by weight.
- the mixture is then incubated at a temperature between about 4° C. and about 8° C., preferably about 4° C., for a period of time to assure that the precipitation is complete enough to serve for analytical purposes. It has been found that the precipitation is complete enough for quantitative assay after about 3-4 hours; however, the incubation can be continued for as long as 18 hours if desired.
- the polyethylene glycol used in the precipitation step should have a molecular weight such that it is capable of inducing precipitation of the CIC-SPA-label complex.
- a suitable polyethylene glycol is one having a molecular weight between about 6000 and about 8000.
- Preferably polyethylene glycol having an average molecular weight of about 6000 (PEG-6000) is used
- the precipitated CIC-SPA-label complex is then washed, e.g., with an aqueous solution of polyethylene glycol, to remove all traces of the serum and unreacted SPA-label.
- the measurement of the amount of label present in the CIC-SPA-label complex is conducted by a technique appropriate to the type of label used. For example, if a radioactive label has been attached to the SPA the amount of radiolabel in the precipitate can be determined by direct counting of radioactive decays per unit time in the conventional manner. If a fluorescent label has been used, the fluorescence of the precipitated material is determined, usually by resuspending the precipitate and measuring the fluorescent intensity in a spectrofluorometer.
- a preferred label is an enzyme label.
- the CIC-SPA-enzyme complex is then contacted with a substrate for the enzyme, e.g., by resuspending the precipitated CIC-SPA-enzyme complex in an aqueous solution of the enzyme substrate. Enzymatic conversion of the substrate is allowed to proceed for a predetermined period of time and the reaction is then terminated. The amount of substrate converted is then measured to determine the amount of CIC-SPA-enzyme complex present.
- a standard series of sera having known amounts of CIC or functionally equivalent material i.e., a material which undergoes the same reaction as CIC with SPA, is run to establish a calibration curve for the assay under particular conditions.
- a plot of concentration versus optical absorbance is made for the series of standards and a smooth curve is drawn through the plotted data points.
- the sample sera are then assayed under the same conditions and the concentration of CIC in the serum is determined by comparison with the calibration curve, as is well known to those skilled in the art.
- an aggregated IgG may be used. This material binds to SPA just as CIC and is therefore functionally equivalent. It is convenient to prepare such a standard by heat aggregation of IgG, e.g., by heating a solution of IgG to a temperature of 62° C.
- An artificial immune complex standard can also be prepared by reacting an antigen with an antibody IgG to the antigen to form an immune complex.
- RA immune complex diseases
- SLE immune complex diseases
- the enzyme used in the assay of the preferred embodiment of this invention may be any enzyme which can convert a substrate to provide a detectable amount of substrate conversion.
- Preferred enzymes are those which can react with a substrate to produce a colored substance or a substance which can react to yield a colored substance which can then be quantitated by conventional absorption spectrophotometry.
- Suitable enzymes include alkaline phosphatase, galactosidase, glucose oxidase, horeseradish peroxidase and the like.
- a preferred enzyme is horseradish peroxidase.
- the enzyme is conjugated to the SPA by conventional procedures, e.g., by treatment of the SPA with glutaraldehyde followed by reaction with the enzyme. Typical conjugation procedures are disclosed in Avrameas, S., Immunochemistry 6, 43-52.
- the substrate used may be an aqueous solution of hydrogen peroxide containing 2,2'-azino-di-(3-ethylbenzothiazoline sulfonic acid) (ABTS).
- ABTS 2,2'-azino-di-(3-ethylbenzothiazoline sulfonic acid
- This mixture develops a color which can be quantitated by measuring the absorption of the solution at a wavelength of 410 nm (A 410 ).
- the serum containing CIC in the form of a diluted serum, e.g., in a 1:40 solution, as is conventional in assays of this type.
- the serum it is also possible to add the serum to be assayed directly to the reagent without preliminary dilution. This procedure further simplifies the assay of this invention.
- the formation of the CIC-SPA-label complex and its precipitation from the serum can be performed in a single step.
- an solution of SPA-label and polyethylene glycol is mixed with the serum containing CIC and incubated for five to fifteen minutes at a temperature of about 18° C. to 25° C. followed by incubation for three to four hours at a temperature of about 4° C. to about 8° C.
- the mixture is incubated for about 15 minutes at room temperature followed by incubation for 3-4 hours at about 4° C.
- the CIC-SPA-label complex forms and is precipitated.
- the amount of label present in the precipitate is determined. For example, if an enzyme label is used, the precipitate is incubated with a solution of the enzyme substrate for a predetermined period of time and the amount of enzymatic conversion which takes place is determined as discussed above.
- the preparation of this solution is somewhat critical.
- Example 1 below provides directions for preparing this reagent for optimum effectiveness.
- This example illustrates the preparation of the reagent used in the preferred embodiment of this invention.
- a solution was prepared by mixing the following ingredients:
- the concentration of the SPA-peroxidase solution to be used is determined by titrating the product of the SPA-horseradish peroxidase conjugation reaction, and adjusting the concentration of the solution used in preparing the reagent so that the volume of reagent used in the assay contains an excess of SPA-peroxidase.
- This example illustrates the analysis of CIC in serum using the preferred embodiment of the method of this invention.
- Immune complexes to serve as standards were prepared by incubation of human albumin (1:8 dilution of 20 mg/ml) and anti-human albumin (Cappel Laboratories) overnight at 4° C. Following the overnight incubation, the solution was centrifuged at 2600 RPM for 20 minutes at room temperature to remove any precipitated protein.
- a series of 12 ⁇ 75 mm glass test tubes was prepared for the standard curve, a blank and a sample as follows: one tube for the blank, five standards labeled 40, 20, 10, 5 and 2.5 micrograms equivalent per milliliter, and one tube for the sample.
- One hundred microliters of diluted standard were pipetted into the tubes labeled 40 and 20 micrograms equivalent per milliliter. Serial doubling dilutions were performed by transferring 100 microliters of solution from standard tube 20 to standard tube 10, mixing, transferring 100 microliters from standard tube 10 to standard tube 5, mixing, transferring 100 microliters of solution from standard tube 5 to standard tube 2.5, mixing and, finally, discarding 100 microliters from standard tube 2.5.
- Example 1 Into each tube was pipetted 0.9 ml of the reagent of Example 1 comprising a solution of protein-A conjugated to horseradish peroxidase in a 3.5% aqueous solution of polyethylene glycol having an average molecular weight of 6000 (PEG-6000). The solutions were mixed well and the reaction mixtures were incubated at room temperature for 15 minutes followed by 3 hours incubation at 4° C.
- PEG-6000 polyethylene glycol having an average molecular weight of 6000
- the tubes were centrifuged at 800 ⁇ g for 30 minutes, the supernatant was carefully decanted and the tubes were inverted and blotted on absorbent paper to remove excess fluid, using great care to avoid disrupting the pellet at the bottom of the tube.
- the pellet was washed twice by mixing with 1.5 ml of wash solution at room temperature, centrifuging at 800 ⁇ g for 30 minutes and carefully decanting the supernatant and drying as described above.
- the absorbance at 410 nm (A 410 ) is plotted on arithmetic graph paper versus the concentration for all the standards.
- the concentration of the immune complex in the sample can then be determined by reading off the plot the concentration of immune complex which gives the measured A 410 .
- This value when multiplied by 40 (the degree of dilution) yields the value of micrograms equivalent per milliliter of aggregated IgG in the serum sample.
- This example illustrates the binding of protein-A-peroxidase to native and heat aggregated IgG.
- IgG human
- a solution of IgG was heat aggregated at 62° C. and then fractionated by exclusion chromatography on a polysaccharide gel column (Sephadex G-200, manufactured by Pharmacia AB) to separate native and aggregated IgG.
- Each fraction was concentrated to the same protein concentration and serial dilutions were made to provide a series of samples having varying concentrations of aggregated and native IgG as indicated in Table 1 below.
- Each of the samples was analyzed by the procedure of Example 2 and the optical absorbance at 410 nm is tabulated in Table 1.
- results as shown in the table indicate preferential binding of the protein-A-peroxidase to the heat aggregated IgG rather than to the native (non-denatured) IgG.
- This example illustrates the preparation of an alternative standard material using artificially prepared immune complexes.
- Immune complexes were prepared by incubation of human albumin (1:8 dilution of a 20 mg/ml solution) and anti-human albumin (supplied by Cappel Laboratories) overnight at 4° C. Following the overnight incubation, the solution was centrifuged at 2600 RPM for 20 minutes at room temperature to remove precipitated protein. Serial doubling dilutions of the supernatant were made to yield a series of samples having varied concentration of the immune complex as listed in Table 2 below.
- the reagents for conducting the immunoassay of the invention may conveniently be supplied to the analyst in the form of a kit for assaying circulating immune complexes in mammalian serum comprising
- the reagents may be supplied in form of solutions of appropriate concentration either for dilution or for direct use in performing the assay.
- the reagents may also be supplied in the form of solids ready for dissolving by adding water or a buffer to the containers.
- the SPA-label and the polyethylene glycol may be combined in one container either in form of solids ready for dissolving or in the form of a solution ready for dilution or for direct use in the assay.
Abstract
Description
TABLE 1 ______________________________________ Protein A.sub.410 micrograms/ml Aggregated Native ______________________________________ 40 1.128 0.146 20 0.581 0.098 10 0.284 0.033 5 0.120 0.010 2.5 0.022 0.000 ______________________________________
TABLE 2 ______________________________________ A.sub.405 Dilution (mean determinations) ______________________________________ 1:1 1.488 1:2 1.034 1:4 0.513 1:8 0.205 1:16 0.086 1:32 0.038 1:64 0.019 Control 0.001 ______________________________________
Claims (21)
Priority Applications (1)
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US06/516,465 US4617262A (en) | 1983-07-22 | 1983-07-22 | Assaying for circulating immune complexes with labeled protein A |
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US06/516,465 US4617262A (en) | 1983-07-22 | 1983-07-22 | Assaying for circulating immune complexes with labeled protein A |
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US4617262A true US4617262A (en) | 1986-10-14 |
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Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4757002A (en) * | 1983-06-13 | 1988-07-12 | Regents Of The University Of Minnesota | Method and kit for the estimation of serum immunoglobulin |
WO1989009628A1 (en) * | 1988-04-04 | 1989-10-19 | Potempa Lawrence A | Binding of immune complexes by modified forms of c-reactive protein |
US4948725A (en) * | 1987-12-10 | 1990-08-14 | University Of Florida Research Foundation, Inc. | Novel type VI bacterial Fc receptors |
EP0398292A2 (en) * | 1989-05-19 | 1990-11-22 | Eisai Co., Ltd. | Diagnostic composition for rheumatoid arthritis |
US4977082A (en) * | 1987-12-10 | 1990-12-11 | University Of Florida Research Foundation, Inc. | Type VI bacterial FC receptors |
US5085984A (en) * | 1987-12-10 | 1992-02-04 | University Of Florida Research Foundation, Inc. | Novel type VI bacterial Fc receptors |
US5112770A (en) * | 1988-06-08 | 1992-05-12 | North Carolina State University | Precipitation of multivalent antiligands with affinity surfactants |
US5122112A (en) * | 1986-11-21 | 1992-06-16 | Imre Corporation | Antigen-specific removal of circulating immune complexes |
US5167925A (en) * | 1988-06-08 | 1992-12-01 | North Carolina State University | Precipitation of multivalent antiligands with affinity surfactants |
US5593897A (en) * | 1988-04-04 | 1997-01-14 | Northwestern University | Binding of immune complexes by modified forms of C-reactive protein |
US20140121125A1 (en) * | 2012-10-11 | 2014-05-01 | Abaxis, Inc. | Peptides, devices, and methods for the detection of ehrlichia antibodies |
US9851352B2 (en) | 2014-04-04 | 2017-12-26 | Abaxis, Inc. | Compositions and methods for identifying Ehrlichia species |
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US3850798A (en) * | 1972-11-06 | 1974-11-26 | J Sjoquist | Method for separating a polypeptide from microorganisms |
US3995018A (en) * | 1972-11-06 | 1976-11-30 | Pharmacia Aktiebolag | Method of binding immunoglobulin employing a polypeptide from microorganisms |
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1983
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US3850798A (en) * | 1972-11-06 | 1974-11-26 | J Sjoquist | Method for separating a polypeptide from microorganisms |
US3995018A (en) * | 1972-11-06 | 1976-11-30 | Pharmacia Aktiebolag | Method of binding immunoglobulin employing a polypeptide from microorganisms |
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Non-Patent Citations (10)
Title |
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Faiferman, I., et al., "Staphylococcal Protein A Fluoroimmunoassay . . . ," Arthritis and Rheumatism 25 (7), 799-801 (1982). |
Faiferman, I., et al., Staphylococcal Protein A Fluoroimmunoassay . . . , Arthritis and Rheumatism 25 (7), 799 801 (1982). * |
Hallgren, R., et al., "Detection of Circulating IgG Aggregates . . ." Ann. rheum. Dis. 35, 306-313 (1976). |
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Cited By (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4757002A (en) * | 1983-06-13 | 1988-07-12 | Regents Of The University Of Minnesota | Method and kit for the estimation of serum immunoglobulin |
US5122112A (en) * | 1986-11-21 | 1992-06-16 | Imre Corporation | Antigen-specific removal of circulating immune complexes |
US4948725A (en) * | 1987-12-10 | 1990-08-14 | University Of Florida Research Foundation, Inc. | Novel type VI bacterial Fc receptors |
US4977082A (en) * | 1987-12-10 | 1990-12-11 | University Of Florida Research Foundation, Inc. | Type VI bacterial FC receptors |
US5085984A (en) * | 1987-12-10 | 1992-02-04 | University Of Florida Research Foundation, Inc. | Novel type VI bacterial Fc receptors |
AU633488B2 (en) * | 1988-04-04 | 1993-02-04 | Northwestern University | Binding of immune complexes by modified forms of c-reactive protein |
WO1989009628A1 (en) * | 1988-04-04 | 1989-10-19 | Potempa Lawrence A | Binding of immune complexes by modified forms of c-reactive protein |
US5593897A (en) * | 1988-04-04 | 1997-01-14 | Northwestern University | Binding of immune complexes by modified forms of C-reactive protein |
US5112770A (en) * | 1988-06-08 | 1992-05-12 | North Carolina State University | Precipitation of multivalent antiligands with affinity surfactants |
US5167925A (en) * | 1988-06-08 | 1992-12-01 | North Carolina State University | Precipitation of multivalent antiligands with affinity surfactants |
EP0398292A3 (en) * | 1989-05-19 | 1991-11-13 | Eisai Co., Ltd. | Diagnostic composition for rheumatoid arthritis |
EP0398292A2 (en) * | 1989-05-19 | 1990-11-22 | Eisai Co., Ltd. | Diagnostic composition for rheumatoid arthritis |
CN1036156C (en) * | 1989-05-19 | 1997-10-15 | 卫材株式会社 | Diagnostic drug for rheumatoid arthritis |
US20140121125A1 (en) * | 2012-10-11 | 2014-05-01 | Abaxis, Inc. | Peptides, devices, and methods for the detection of ehrlichia antibodies |
US9651546B2 (en) * | 2012-10-11 | 2017-05-16 | Abaxis, Inc. | Peptides, devices, and methods for the detection of ehrlichia antibodies |
US9696300B2 (en) | 2012-10-11 | 2017-07-04 | Abaxis, Inc. | Peptides, devices, and methods for the detection of ehrlichia antibodies |
US10444231B2 (en) | 2012-10-11 | 2019-10-15 | Abaxis, Inc. | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
US10948487B2 (en) | 2012-10-11 | 2021-03-16 | Zoetis Services Llc | Peptides, devices, and methods for the detection of Ehrlichia antibodies |
US9851352B2 (en) | 2014-04-04 | 2017-12-26 | Abaxis, Inc. | Compositions and methods for identifying Ehrlichia species |
US11204351B2 (en) | 2014-04-04 | 2021-12-21 | Zoetis Services Llc | Compositions and methods for identifying Ehrlichia species |
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